Identification Of Amino Acids Content Natural Variation And Associated Genes In Camellia Sinensis

Tea plants [Camellia sinensis(L.)O.Kuntze] is an important economic crop,and its fresh leaves are riched in amino acids.Free amino acids make much contribution to the flavor of tea,so that systematic study of amino acids in tea plants are significant.To date,there are few studies focusing on amino acid association analysis,functional genes mining,and the molecular regulation mechanism of amino acid is unknown.In this study,liquid chromatography-mass spectrometry system was performed to measure amino acids content in fresh leaves(young leaves,mature leaves)of nine tea cultivars,which were collected from representative geographical origins.The content of 12 amino acids in nine tea cultivars were obtained.Combined with the transcriptome data,correlation network between DEGs and amino acids was constructed.The differences of gene expression and amino acid content between tissues and several tea cultivars were analyzed.In addition,this study measured the content of theanine and proline in young leaves of 174 tea accessions.Combined with the genotype data of the population,genome-wide association studies were performed.132 quantitative trait loci(QTLs)were identified.A total of 713 genes were annotated within these QTLs regions.The candidate gene as CsFPGS was selected for further function analysis.The main results are as follows:1.Metabolites in two tissues of nine tea cultivars(‘Zijuan’,‘Qianmei 502’,‘Echa10’,‘Anjibaicha’,‘Fudingdabai’,‘Tieguanyin’,‘Wuniuzao’,‘Enchahong’,‘Huangyan’)were measured by using UPLC-MS.A total of 12 amino acids(valine,tryptophan,threonine,proline,phenylalanine,arginine,leucine,glutamic acid,theanine,aspartic acid,glutamine and 2-Hydroxyphenylalanine)were identified.Based on the transcriptome data,a total of626 differentially expressed genes and 20 transcription factors were identified between mature leaves and young leaves.2.The level of theanine and proline varied at a large extent in fresh leaves of 174 tea accessions.According to the statistic result,the content of theanine and proline have variations in two environments(July,2018 and April,2019).The coefficient ranges of theanine and proline are 40.22%-57.29% and 30.43%-32.95%,respectively.Based on the theanine and proline content and genotype data in two environments of 174 tea plants,genome-wide association study was performed.In total,132 QTLs were identified to be associated with theanine and proline contents(-log10(P-value)> 3).713 genes were identified within these QTLs regions.In the end,CsFPGS was selected for alleles diversity analysis and functional research.3.Subcellular location result showed that CsFPGS was located in the chloroplast.Over-expression of CsFPGS in tobacco leaves induced the accumulation of proline,glutamic acid and glutamine in tobacco leaves.Similar phenotypes were observed in transgenic Arabidopsis.These results confirmed that CsFPGS is a key gene regulating the accumulation of free amino acids(theanine,proline et al)in tea plants.In summary,this study lays a foundation for studying the genetic variation of theanine and proline in tea plants.It provides abundant genetic resources for elucidating the molecular mechanism of theanine and proline content variation in tea plants.